RESUMO
Because RNAs fold as they are being synthesized, their transcription rate can affect their folding. Here, we report the results of single-molecule fluorescence studies that characterize the ligand-dependent cotranscriptional folding of the Escherichia coli thiM riboswitch that regulates translation. We found that the riboswitch aptamer folds into the "off" conformation independent of its ligand, but switches to the "on" conformation during transcriptional pausing near the translational start codon. Ligand binding maintains the riboswitch in the off conformation during transcriptional pauses. We expect our assay will permit the controlled study of the two main physical mechanisms that regulate cotranscriptional folding: transcriptional pausing and transcriptional speed.
Assuntos
Dobramento de RNA , Riboswitch , Tiamina Pirofosfato/metabolismo , Transcrição Gênica , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Tiamina Pirofosfato/químicaRESUMO
Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway). We employed an optical trapping assay to probe the motions of individual RNAP molecules transcribing a DNA template carrying tandem repeats encoding the his pause, subjecting these enzymes to controlled forces. NusA significantly decreased the pause-free elongation rate of RNAP while increasing the probability of entry into short- and long-lifetime pauses, in a manner equivalent to exerting a ~19 pN force opposing transcription. The effects of force and NusA on pause probabilities and lifetimes support a reaction scheme where nonbacktracked, elemental pauses branch off the elongation pathway from the pretranslocated state of RNAP.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Pinças Ópticas , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Cinética , Fenômenos Ópticos , Fatores de Alongamento de Peptídeos/genética , Plasmídeos , Sequências de Repetição em Tandem , Moldes Genéticos , Fatores de Transcrição/genética , Fatores de Elongação da TranscriçãoRESUMO
Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Transcrição Gênica , Carbocianinas/química , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Corantes Fluorescentes/química , Regiões Promotoras Genéticas , Fator sigma/química , Fator sigma/genética , Fator sigma/metabolismo , Imagem Individual de Molécula , Regiões Terminadoras Genéticas , Iniciação da Transcrição Genética , Terminação da Transcrição GenéticaRESUMO
NusA is a core, multidomain regulator of transcript elongation in bacteria and archaea. Bacterial NusA interacts with elongating complexes and the nascent RNA transcript in ways that stimulate pausing and termination but that can be switched to antipausing and antitermination by other accessory proteins. This regulatory complexity of NusA likely depends on its multidomain structure, but it remains unclear which NusA domains possess which regulatory activity and how they interact with elongating RNA polymerase. We used a series of truncated NusA proteins to measure the effect of the NusA domains on transcriptional pausing and termination. We find that the N-terminal domain (NTD) of NusA is necessary and sufficient for enhancement of transcriptional pausing and that the other NusA domains contribute to NusA binding to elongating complexes. Stimulation of intrinsic termination requires higher concentrations of NusA and involves both the NTD and other NusA domains. Using a tethered chemical protease in addition to protein-RNA cross-linking, we show that the NusA NTD contacts the RNA exit channel of RNA polymerase. Finally, we report evidence that the NusA NTD recognizes duplex RNA in the RNA exit channel.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/fisiologia , Fatores de Alongamento de Peptídeos/fisiologia , RNA Bacteriano/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Conformação Proteica , RNA Bacteriano/química , Regiões Terminadoras Genéticas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Elongação da TranscriçãoRESUMO
We have identified minimal nucleic acid scaffolds capable of reconstituting hairpin-stabilized paused transcription complexes when incubated with RNAP either directly or in a limited step reconstitution assay. Direct reconstitution was achieved using a 29-nucleotide (nt) RNA whose 3'-proximal 9-10 nt pair to template DNA within an 11-nt noncomplementary bubble of a 39-bp duplex DNA; the 5'-proximal 18 nt of RNA forms the his pause RNA hairpin. Limited-step reconstitution was achieved on the same DNAs using a 27-nt RNA that can be 3'-labeled during reconstitution and then extended 2 nt past the pause site to assay transcriptional pausing. Paused complexes formed by either method recapitulated key features of a promoter-initiated, hairpin-stabilized paused complex, including a slow rate of pause escape, resistance to transcript cleavage and pyrophosphorolysis, and enhancement of pausing by the elongation factor NusA. These findings establish that RNA upstream from the pause hairpin and pyrophosphate are not essential for pausing and for NusA action. Reconstitution of the his paused transcription complex provides a valuable tool for future studies of protein-nucleic interactions involved in transcriptional pausing.